Our proposed research focuses on defining the mechanism of action of the TlpA and TlpB chemoreceptors that play fundamental roles in pathogenesis of the ulcer-causing bacterium Helicobacter pylori. These receptors modulate H. pylori-induced inflammation. There is a fundamental gap, however in our understanding of the TlpA and TlpB signals, of how these receptors sense their ligands, how they perform signal transduction, and how they promote pathogenesis. Continued existence of this gap prevents us from gaining a full understanding of H. pylori's pathogenic mechanisms and, in the long term, thwarting these processes to enable the creation of new drugs against H. pylori-related disease. Millions of people worldwide and in the U.S. are infected by H. pylori and suffer from its associated diseases-ulcers and gastric cancer. Gastric cancer is the second cause of cancer deaths worldwide. H. pylori is here to stay based on recent studies that show H. pylori incidence has stabilized in the developed world. Furthermore, current therapies to cure H. pylori infection fail with unacceptable frequency, e.g., recent estimates in the United States have found that 20-25% of infected individuals are not cured by the current therapeutic regime. New drug targets are desperately needed. The specific objective of this application is to dissect TlpA and TlpB signal transduction and its role in gastric disease. Our central hypothesis is that TlpA and TlpB transduce information from specific ligands to affect bacterial-host interactions, proinflammatory gene expression, and in turn, host inflammation. Our hypothesis has been formulated from preliminary data using small molecule arrays to identify specific new ligands of TlpA, developing key reagents for analyzing TlpA and TlpB signal transduction, defining the roles of TlpA and TlpB in inflammation, and developing new assays for analyzing H. pylori in the gastric setting. In Aim 1, we determine the mechanism of TlpA and TlpB ligand binding and signal transduction, using in vitro binding assays with purified protein, in vitro chemotaxis signal transduction assays, and intact H. pylori chemotaxis assays. In Aim 2, we determine the mechanism by which TlpA and TlpB modulate inflammation, analyzing the nature of the inflammatory response provoked by tlpA or tlpB mutants. In addition, we will explore the possibility that TlpA and TlpB mutants have abnormal host cell interactions and distribution. Lastly, we investigate the hypothesis that host parameters change of the course of an infection using metabolomics to analyze nutrient distribution in the stomach and state-of-the art organoid culture systems. The proposed research is innovative in that it will create new knowledge about the functions of chemotaxis during bacterial pathogenesis, and it its use of state of the art approaches. The proposed research is significant because it addresses chemotaxis, a fundamental property important to many pathogens, and focuses specifically on a bacterium that causes rampant disease but for which we are losing antibiotic efficacy. The long-term outcomes generated by this research are likely to provide insights that will enable creation of new drugs against H. pylori-related disease.